Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. The unusually high number of serine, leucine, and arginine residues in secretin has precluded the use of oligonucleotides to screen cDNA libraries to isolate a secretin cDNA. In the present study, a short cDNA encoding porcine secretin was amplified from duodenal mucosal first-strand cDNA template by using 16,384- and 4096-fold degenerate primers in the DNA polymerase chain reaction. From the sequence of the amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a cDNA library. Here we report the sequences of cDNAs encoding the porcine and rat secretin precursors. The predicted amino acid sequences reveal that each precursor consists of a signal peptide, an N-terminal peptide, secretin, and a 72-amino acid C-terminal peptide. Secretin has been highly conserved through evolution. Rat secretin differs from its porcine counterpart by a single glutamine-for-arginine substitution at position 14. In contrast, the amino acid sequences of the C-terminal peptides are only 39% conserved between the two species, suggesting that the C-terminal peptide does not have an essential physiologic function. RNA blot hybridizations reveal that the rat secretin gene is expressed throughout the small intestine. Although secretin immunoreactivity has been localized in the central nervous system by some laboratories, we are unable to detect secretin mRNA in tissues of the central nervous system by Northern blot hybridization.